JPS61212289A - Novel antibiotic inarmycin and production thereof - Google Patents

Abstract

NEW MATERIAL:Antibiotic inarmycin. Appearance: hygroscopic white powder; molecular formula: C27H43N3O13S; color reaction: positive in ninhydrin reaction, 2,4-dinitrophenylhydrazine reaction, Elson-Morgan reaction; solubility: soluble in water and methanol, slightly soluble in acetone and ethyl acetate; having infrared absorption spectrum and ultraviolet absorption spectrum shown by the figures. USE:A carcinostatic agent inhibiting growth of human leukemia K562 and MOLT-3 cell, mouse leukemia L1210 cell, Lewis lung cancer cell, mouse lymphoma L5178Y cell, its ACRUBICIN resistant strain and bleomycin resistant strain. PREPARATION:A bacterium [e.g., IM8251 T strain (FERM P 8137)] belonging to the genus Streptomyces, capable of producing inarmycin, is cultivated, inarmycin or its salt is accumulated and inarmycin or its salt is collected from the culture mixture.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a salt thereof and a method for producing the same. More specifically, the present invention relates to a new antibiotic inalmycin or a salt thereof obtained by culturing actinomycetes belonging to the genus Streptomyces, and a method for producing the same.

As a result of searching for new and useful antibiotics, the present inventor discovered that a new antibiotic, inalmycin, or its salts, can be produced fermentatively by culturing actinomycetes belonging to the genus Streptomyces in a nutrient medium. , isolating the antibiotic;
Its physicochemical and biological properties were determined. The present invention has been completed based on the above findings.

Therefore, the present invention has the following properties: it is a hygroscopic white powder, and its molecular formula is C27H4,N3O.
13S, and the infrared absorption spectra measured with odor f-hypotium tablets are shown in Figure 1. As shown in Figure 2, it is ninhydrin reaction, λ, Hiro-dinitrophenylhydrazine reaction and B I son-Morgan
The present invention provides a new antibiotic, inalmycin, and its salts, which are positive in the reaction, soluble in water and methanol, and sparingly soluble in acetone and ethyl acetate.

The new antibiotic inalmycin is also 044? , lJ section, H7, /μchi, 03ko, / /%, N+, gr%,
s≠,! /C27H43N3O
Theoretical value of 13S (C≠ Ta, Ta / Chi, H4,47
Chi, 03 ko, 0/regret.

NJ, ≠ 7%, Sμ, 23 Chino, and this molecular formula is in good agreement with the FAR mass spectrum of inalmycin ((M+H
) : m/e 6 j O) proved by n.

Furthermore, the H and 13O nuclear magnetic resonance spectra measured in heavy water are as shown in Figure @3 and Figure μ, respectively, and the ultraviolet absorption curve of the i-th covele is in water and 0.0/N.
23 in sodium hydroxide! nm(E”==/lj
) each showed an absorption maximum.

/crn From the above-mentioned physicochemical properties, inalmycin was clearly distinguished from known antibiotics, and was confirmed to be a new antibiotic.

Inalmycin can also be produced in salt form,
Typical salts include salts with inorganic and organic acids, such as hydrochlorides, sulfates, phosphates, acetates, etc., and metal salts, such as alkali metal salts and alkaline earth metal salts, especially sodium salts. I can do it.

The minimum inhibitory concentrations of inalmycin against various types of bacteria on heart infusion agar medium and against various fungi on yeast glucose agar medium are shown in Table 1-(
As shown in 1) and 0), it exhibits almost no antibacterial activity at a concentration of iooβri/at.

Table l-(7) Z Staphylococcus aureus rAM101/
〉1002 Micrococcus luteus IAM
#), tj 〉10゜3, Bacillus subtilis POIJ/ta >io.

Gupatillus cereus T
>io.

j Bacillus megaterium IAM/(7,1>100
tCorynebacterium xerosis
)/DO7O7 Mycobacterium megmatis R/j
〉1001 Mycobacterium Frey 1sota')to.

Taesierihia coli B
>100io, Escherichia Cori/Co
〉1001/, Pseudomonas aeruginosa IFO34L1! >10012.
Pseudomonas Fist Fluorescence Hj)
/ 00i3. salmonella typhi
>io.

Salmonella enteritidis //
〉1001 Shigella Sonay
>io.

/J, Proteus vulgaris
>io.

(Note) Medium Nihard Infusion Agar Medium Culture Temperature = 37℃ Table 1) Yourspergillus oryzae IkM2tJO〉1003 Juice? Tritis cinerea IhMzi2&
>10゜Hiromolcellara Lamanianus IkMt/21
〉100 Tabenicillium chrysogenum IAM
7jko4 ＞/(70t, Candida albicans 〉'''7tJ7jidar'
)-1-1) SuY21-4 〉/DO
r Cryptococcus neoformans IAM/Jjyo3
>io.

Tasatsucharomyces cerevisiae YjJ-2>io.

(Medium used: yeast glucose agar culture temperature = 27°C Inalmycin Bio4 fetal bovine serum added R, PM I-/
Human leukemia Kj4j and MOL T in JμO medium
-3 cells, leukemia L/co10 cells and Lewis lung cancer cells in mice. In addition, IO% horse serum addition 11
Mouse synosoma L in PMI-/4g0 medium
Completely inhibits the growth of zi71Y1m cells and their acurubicin-resistant and pleomycin-resistant strains. The growth inhibition and concentration of Ochi against these cancer cells are shown in Table 2.
o, r -o, otsu3μ? It is within the range of selection.

Table 2 Human leukemia Kjt2 cells O, Koihito leukemia MOLT-J cells θ! /Mouse leukemia L/Leukemia O, Kohiro Mouse lymphoma Lj/
71' (Cell 032 is the acurubicin-resistant strain as above. 0.20 is the pleomycin-resistant strain as above.
0.2/mouse Law is lung cancer cells
0.1. J (Note) Medium used: PRMI −/
luo medium, 10 oes horse serum added to Lri7rYMI cells, and 1 oes fetal bovine serum added to other cells.

Culture: 37°C, 72 hours Inalmycin shows no acute toxicity even if 200 q/ml is injected intravenously or intraperitoneally into mice.

Therefore, inalmycin is expected to be an anticancer drug.

The second gist of the present invention is to culture inalmycin-producing bacteria belonging to the genus Streptomyces to accumulate inalmycin or its salt, and to collect inalmycin or its salt from the culture. It's in the method of manufacturing salt. According to the invention, inalmycin or a salt thereof is obtained in pure or crude form, in solution or in solid form.

An example of an inalmycin-producing bacterium is the nycWJ strain, which is an actinomycete isolated from the soil of Inariyama, Omuta City, Fukuoka Prefecture in May 1980, and is designated by the strain number IMrcoj/T. IM! The mycological properties of the r2 t/'r strain are as follows.

l. Morphology Under the microscope, the IMr coj/T strain forms slightly curved aerial hyphae that are relatively longer than branched basal hyphae, and the tips of the aerial hyphae are straight and slightly curved. Whorled branches are not allowed. Mature spore e has a chain of 70 or more spores, and the spore sizes are o, r~o, + x o, t~
The surface of the spore is smooth at about 0,0 micrometers.

2. Growth status on various media (27°C, 2 weeks culture) Good growth on sucrose/nitrate agar medium. Yellow powdery aerial mycelium? It grows epiphytically, and no soluble pigment is observed.

G27. Growth on glucose-asparagine agar medium was normal. It grows yellow-brown powdery aerial mycelium, and no soluble pigments are observed.

0J glycerin-asparagine agar medium (ISP-
Medium) Growth is good. It grows on slightly greenish-gray velvety aerial mycelium, and no soluble pigments are observed.

(Hiro) Nutachi jar inorganic salt agar medium (ISF-medium 4t
) Good growth. It grows yellow-gray velvety aerial mycelium, and no soluble pigments are observed.

(Lt) Good growth on tyrosine agar medium (ISP-Medium 7). It grows yellow velvety aerial mycelium, and no soluble pigment is observed.

(,<) Growth on nutrient agar medium was normal. It grows on slightly reddish white velvety aerial mycelium, and no soluble pigments are observed.

(7) Good growth on yeast/malt agar medium (ISP-medium-2). 11. Yellow velvety aerial mycelia were grown, and no soluble pigment was observed. do not have.

V> Good growth on oatmeal agar medium (ISP-Medium 3). It grows yellow powdery aerial mycelia, slightly visible on the brown soluble pigment.

3. Physiological properties (1) Liquefaction of gelatin In glucose-peptone-gelatin medium (core °C culture), liquefaction begins around the 7th day after cultivation, and its effect is moderate.

(2) Starch hydrolysis (starch/inorganic salt agar medium, cultured at 27°C) Hydrolyzability was observed from around 7 days after culture, and its effect was moderate.

(3) Coagulation of skim milk - peptonization (skim milk, 27℃
Culture) After culture, no obvious coagulation was observed, but peptonization was observed around 100 days after culture, and its effect was somewhat strong.

Production of melanin-like pigment (Tyrosine agar (ISP-
Medium 7, -27°C culture) No production of melanin-like pigment was observed.

(j) Availability of carbon source (Pridham-Gotlieb agar medium 15P-medium, cultured at 27°C) D-Xylose% D-
It grows well using mannitol, D-7 lactose, rhamnose, and D-glucose. Inositol and raffinose can also be used, but the growth is not so good. L-
Arabinose and sucrose are hardly available.

(J) Growth temperature range (yeast/malt agar l5P-
Medium λ, oatmil agar 15P-medium 3, λ weeks) Grows at 10-37°C.

To summarize the above characteristics, the IMlr, 2jlT strains belong to the genus Streptomyces, and L-type diaminopimelic acid [:LL-DAPI was detected in the whole bacterial cell components of this strain.The inlet hyphae are straight. It is slightly curved, no whorls are visible, and the surface of the spores is smooth. In various media, aerial mycelium often appears yellow and produces little soluble and melanin-like pigments. Regarding proteolytic ability, coagulation of skim milk is extremely weak, but peptonization of skim milk and liquefaction of gelatin are moderate. In addition, the hydrolyzability of starch is moderate. L-arabinose and sucrose are hardly used, but D-xylose, inositol, D-mannitol, D-7 lactose, rhamnose, raffinose, D
- Utilize glucose.

Looking at the above characteristics, Streptomyces glius (
Streptomyces griseus IM
I thought about naming lr2 and lT stock.

This strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology/110
After March 71, FERM
It has been deposited as P-4/37).

As seen in other actinomycetes strains, the IMrco J-/T strain is susceptible to changes in its properties and cannot be mutated by artificial mutagenic means using, for example, ultraviolet rays, X-rays, radiation, or chemicals. Any mutant strain capable of producing inalmycin can be used in the method of the present invention, even if it is a mutant strain.

In carrying out the method for producing inalmycin of the present invention, the inalmycin producing strain Streptomyces griseus IMJ1. The t/T strain was inoculated into a nutrient-containing medium and allowed to grow aerobically (thus, a culture containing inalmycin was obtained. As a nutrient, one known as a nutrient source for actinomycetes was added. For example, commercially available peptone, meat extract, corn staple liquor, cottonseed flour, peanut flour, soybean flour, yeast extract, NZ-amine, thawed casein, fish meal, sodium nitrate, ammonia nitrate,
Nitrogen sources such as ammonia sulfate and commercially available Glyse IJ7% Ill, Ylucose, Maltose,
Carbohydrates such as molasses or carbon sources such as fats and salt,
Inorganic salts such as phosphates, calcium carbonate, magnesium sulfate can be used. In addition, trace amounts of metal salts can also be added if necessary. These items are used by the producing bacteria and are useful for the production of inalmycin, so 6n is enough.
- All known culture materials for actinomycetes can be used. Liquid culture is preferred for mass production of inalmycin;
The culture temperature can be applied within a range that allows the producing bacteria to grow and produce inalmycin. Cultivation is usually continued until sufficient inalmycin has accumulated. For example, oatmeal 2X1
Liquid medium consisting of yeast extract <7.7X (pH 7
, / ) on an agar slant medium to make sure IMI2! / T strain was inoculated and cultured aerobically (twice shaking culture) at 27°C.
Accumulation of inalmycin was observed from day 1 of culture at r17t.

When culturing the actinomycete rW2j/T strain for the production of inarmycin, in addition to the above-mentioned shaking culture, a jar incubator or a large stainless steel tube, which is generally used for the aerated agitation culture of microorganisms, is used. Manufacturing tanks and culture tanks are also used for mass production. In the case of large-scale culture (U is a culture solution of all species cultured in the above-mentioned liquid medium with shaking for about λ days, it is preferable to inoculate fl, fl, !r ~ 3.0% (volume)).

Inalmycin in the culture solution can be collected using various adsorbents or ion exchange resins.

For example, porous adsorption resin Diaion H is used as an adsorbent.
If P-20 or 0HP-, ;tOp is used, HP
Inalmycin adsorbed on -coO or 0HP-20p is eluted and purified with a solvent such as water or methanol.

For example, if DOWEXTA 0 (Na+ type) is used as the ion exchange resin, 0. ! Elute with an acid such as N-hydrochloric acid (2). After concentrating the eluate containing inalmycin, perform high performance liquid chromatography (H
By removing impurities (PLO) method, pure inalmycin can be obtained.

In the high performance liquid chromatography method for inalmycin,
Various fillers can be used, such as c-
Irsilica (Nucleosil! 018 or LLP-/) manufactured by Whatman Co., Ltd. is used.

As a developing solvent, a mixture of methanol, water, acetic acid, acetonitrile, etc. can be used;
IL when a mixture of acetonitrile-0,- and acetic acid is used
Ru.

Inalmycin can be purified in a pure manner by appropriately combining or repeating the above-mentioned extraction, separation, or purification methods.

Examples are shown below, but since the physicochemical properties of inalmycin have been clarified by the present invention, its properties (=
Based on this, all kinds of methods for producing inalmycin can be devised. Therefore, the present invention is not limited to the Examples, and of course, modification means of the Examples may be used. Based on the properties of inalmycin revealed by the present invention, known means may be applied to inalmycin. Covers all methods of production, concentration, extraction, and purification.

Example 200-ml strain MIr-2jlT cultured on agar slant medium was cultured in a liquid medium consisting of oatmeal 2X1 yeast extract/X (pH 7) containing 100 mt f.
The mixture was inoculated into an Erlenmeyer flask and cultured at 27° C. for ≠0 hours on a rotary shaker (iro rotations per minute) to obtain a seed culture.

Next, include a liquid medium rote with the same composition as above.

Seed culture solution (1 Erlenmeyer flask per liter, approx./l) was inoculated into a stainless steel jar incubator with a capacity of λ, and cultured at -27°C for 72 hours (stirring: every minute ≠ 00
rotation, aeration t: every molecule ot).

The culture solution was continuously centrifuged at μ°C to remove bacterial cells. Centrifugation supernatant (pHJ'~6)! After adjusting the pH with N caustic soda, add 3 of Diaion HP20 and stir for 2 hours.
adsorption ratio of active substances. Column with resin: 2 packed, 10
After washing with ion-exchanged water (1), it was eluted with methanol (101), the eluate was concentrated to dryness, and the insoluble matter was separated by replacing it with (500-) water. Adjust the pH of the aqueous solution to r, o (=) and adsorb it on a DOWEX tO (Na+ type)/1 column,
After washing with re-distilled water rt, it was eluted with 3 t of o, rN hydrochloric acid. After neutralizing the eluate with caustic soda, it was concentrated to dryness and dissolved in methanol.
Extracted with O- three times. After filtering the extract, it was concentrated to dryness again, dissolved in 3O- water, and Cellulofine Go-/J'-
Gel filtration is performed at m (ψ≠X r j cm ).

Develop with water, separate /j- fractions, collect the active fractions, and adjust the pH.
rt; After adjustment, OHP'2θP column (ψ/, 6X
/Ion), washed with water and chlorinated)I
Jum was excluded. The active substance was eluted with 10% methanol, and the active fractions were collected and concentrated under reduced pressure. Reversed phase silica gel HPLO' was performed using a preparative column sound (8SQODS 7t2 (KO ψ
10mt/min, holding time/! Minutes. The active fractions of HPLO were collected and freeze-dried to yield approximately 4! OO■ pure inalmycin was obtained.

[Brief explanation of the drawing]

FIG. 4 shows an infrared absorption curve measured with potassium bromide tablets of inalmycin. Figure 2 shows the results in aqueous solution, 0°01·, N hydrochloric acid solution, and 0°.
The ultraviolet absorption curve of inarmycin was measured in a 0IN caustic soda solution. Figure 3 shows IHNMR of inalmycin. wJIA diagram is 13O NMR of inalmycin? show. (1) Complete Deca ring, -) Off-resonance
Decoupling. Figure 2 Wavelength (vm)

Claims (1)

[Claims] 1. It is a hygroscopic white powder with the following characteristics, its molecular formula is C_2_7H_4_3N_3O_1_3S, and the infrared absorption spectrum measured with a potassium bromide tablet is shown in Figure 1. and 0.01 in hydrochloric acid
The ultraviolet absorption spectrum in N sodium hydroxide is the second
As shown in the figure, it is positive for ninhydrin reaction, 2,4-dinitrophenylhydrazine reaction and Elson-Morgan reaction, soluble in water and methanol, acetone,
A new antibiotic, inarmycin, and its salts, which are characterized by being sparingly soluble in ethyl acetate. 2. A method for producing a new antibiotic inalmycin or its salt, which comprises culturing inalmycin-producing bacteria belonging to the genus Streptomyces in a medium containing a nutrient source, and collecting inalmycin or its salt from the culture. .